The long-term goal of the proposed research is to understand the neural mechanisms underlying the control of motor output under normal and pathological conditions. The central hypothesis is that brainstem cholinergic systems contribute to motor preparation in much the same way that they are thought to contribute to sensory attention: by modulating activity in motor structures such that the movements most in line with behavioral goals are most likely to be executed. I examine this hypothesis in a robust anatomical projection of an advantageous animal model system: The cholinergic input from the brainstem pedunculopontine tegmental nucleus (PPT) to a midbrain area, the intermediate gray layer of the superior colliculus (SC) in the mouse. The project goals will be achieved by manipulating (using optogenetics) and recording neural activity in freely-moving transgenic mice to establish how this cholinergic input modulates the activity SC neurons. In a separate set of experiments, behavioral experiments will directly report how PPT signaling modulates motor preparation. Aim 1 will examine how cholinergic input modulates intermediate layer SC activity in vivo. Aim 2 will serve to translate findings in Aim 1 into the behaving mouse by focusing directly on how this cholinergic input alters directional movements. If successful, our proposal will elucidate, in a genetically accessible mouse model, key neural substrates underlying motor preparation. In addition to testing the specific hypotheses proposed here, I will then combine both Aims and directly examine motor-preparatory activity in the behaving mouse during optogenetic manipulation of the cholinergic PPT. All of these experiments make possible future research into how other genetically-defined networks of neurons contribute to motor output. Ultimately, understanding normal motor output can contribute to improving therapies for movement disorders, such as Parkinson's disease.